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. 2022 Apr 8;119(15):e2110256119. doi: 10.1073/pnas.2110256119

Fig. 4.

Fig. 4.

FKBP52 is involved in transcriptional activation of ERα target genes. (A) MCF7 cells expressing shControl or shFKBP52 were cultured in the presence of Dox, and cells were collected and counted. Data are expressed as the mean ± SEM of three independent experiments. *P < 0.05, ****P < 0.0001 by two-tailed Student’s t test. Knockdown efficiency by immunoblotting is shown below the graph. (B) GSEA profiles of estradiol response comparing MCF7 cells expressing shControl and shFKBP52. Sizes, NES (normalized enrichment score), nP (nominal P value), and FDR (false discovery rate) are shown. Three biological replicates were analyzed. (C) T47D cells expressing indicated shRNAs were cultured in medium containing charcoal-stripped serum and Dox for 1 d. The cells were transiently transfected with ERE-luciferase reporter plasmids. After 24 h of transfection, the cells were treated with E2 for another 24 h and luciferase activity was measured. Data are expressed as the mean ± SEM of three independent experiments. **P < 0.01 by two-tailed Student’s t test. (D) T47D cells were cultured in medium containing charcoal-stripped serum for 1 d. The cells were transiently transfected with ERE-luciferase reporter plasmids. After 48 h of transfection, the cells were treated with the indicated concentration of FK506. After 1 h of FK506 treatment, the cells were treated with 10 nM E2 for another 6 h and luciferase activity was measured. Data are expressed as the mean ± SEM of three independent experiments. **P < 0.01, ****P < 0.0001 by Dunnett test. (E) MCF7 cells stably expressing FKBP52 WT, F67D/D68V, or K354A were cultured in medium containing charcoal-stripped serum for 1 d. The cells were transiently transfected with ERE-luciferase reporter plasmids. After 24 h of transfection, the cells were treated with E2 for another 24 h and luciferase activity was measured. The results from three independent experiments are shown. Data are expressed as the mean ± SEM *P < 0.05 by Dunnett test. (F) MCF7 cells expressing shControl or shFKBP52 were transplanted subcutaneously into the flank of NOD/Shi-scid IL-2RγKO mice (n = 7). Tumor size was measured twice weekly. If tumor was undetected, tumor size was considered to be 0. *P < 0.05 by one-tailed Mann–Whitney U test. (G and H) Paraffin-embedded inoculated tumors arising from MCF7 cells expressing shControl (n = 4) or shFKBP52-1 (n = 2) were subjected to immunohistochemical assay using anti–Ki-67 antibody. Representative images of immunohistochemical staining for Ki-67 were obtained using NanoZoomer 2.0RS (G). (Scale bar, 20 μm.) The percentage of Ki-67–positive cells was determined using QuPath, version 0.3.1, software (H). *P < 0.05 by Student’s t test. DMSO, dimethyl sulfoxide.